This invention relates to five pure strains of Rhodococcus erythropolis CNCM I-2204, Rhodococcus erythropolis CNCM I-2205, Rhodococcus erythropolis CNCM I-2207, Rhodococcus erythropolis CNCM I-2208, and Rhodococcus rhodnii CNCM I-2206 that can selectively eliminate organic sulfur from sulfur-containing organic molecules present in certain fossil fuels, and their use in processes for desulfurizing these fuels, in particular petroleum and some of its fractions and their derivatives.
In addition to carbon, fossil fuels such as coal and petroleum contain other elements, such as, for example, sulfur and nitrogen. Thus, crude oil contains sulfur, mainly in organic form, in more or less high concentrations, in general between 0.025% and 5%. This sulfur still remains in a more or less high ratio in the petroleum fractions obtained by distillation (for example up to 10% in certain heavy fractions), and subsequently also in the various petroleum products. In crude oil, sulfur is present mainly in the form of organic sulfur (sulfides, thiols, thiophene, benzothiophene, dibenzothiophene and their substituted derivatives). In several crude oils such as Texas crude, about 70% of the organic sulfur is present in the form of dibenzothiophene (DBT) or alkylated derivatives of DBT.
Combustion of the sulfur present in the fuels causes formation of sulfur dioxides that give rise to acid rain and that are considered to be among the worst air pollutants. In order to limit these noxious emissions, legislation has set standards for sulfur content in petroleum fuels. Moreover, these standards are becoming increasingly strict. For example, the nations of the European Union decided to set the upper limit of gasoil sulfur content at 350 ppm in the year 2000 and it will probably be around 50 ppm in the year 2005. The current specifications for gasoil have been 500 ppm since October 1996, and the preceding specification was 0.2%.
Such specifications dictate accelerated desulfurization of petroleum products by high-performing and economical processes.
Conventional desulfurization processes used in the refining industry implement physico-chemical hydrodesulfurization techniques which allow the reduction of the Cxe2x80x94S bonds in hydrogen sulfide (H2S). This reaction is catalyzed by metallic catalysts and takes place in the presence of hydrogen at a high temperature. The deep desulfurization processes required by the new standards call for working at higher temperatures and at increasingly higher partial hydrogen pressures, and lengthening the dwell time in the reactors. All these factors considerably increase the cost of the desulfurization process. In addition, to attain very low sulfur contents, it is becoming necessary to remove the most refractory sulfur compounds in hydrodesulfurization. To use the example of gasoil, the most refractory compounds are often represented by DBT and its derivatives. It is therefore necessary to increase the hydrogen pressure, the temperature and the dwell time to attain low sulfur contents, thereby considerably increasing operating costs. Moreover, hydrodesulfurization operating conditions can sometimes be so strict that a possible degradation of hydrocarbons other than said sulfur-containing compounds results. Finally, in certain cases, the high concentration of heavy metals in the petroleum may limit the use of hydrodesulfurization catalysts which are sensitive to the presence of the latter. Since the heavy metal concentration and sulfur concentration often increase in a parallel manner during refining, this problem may also limit the implementation of hydrodesulfurization processes.
This is why the development of processes for desulfurization of petroleum or at the very least of some petroleum compounds other than chemical hydrodesulfurization has been studied for several years. This is particularly the case of biological processes that are still called biodesulfurization (BDS) processes.
Several methods of microbiological desulfurization have been described in the literature. Thus, certain sulfate-reducing anaerobic microorganisms are able to degrade DBT with production of H2S. These are slow processes that require reducing elements that may be supplied electrochemically as described in U.S. Pat. No. 4,954,229 or in the form of molecular hydrogen.
There are microorganisms that can aerobically oxidize DBT. In the majority of cases, such systems degrade DBT by using the so-called Kodama metabolic method (Kodama et al., Agr. Biol. Chem., 34, 1320, (1970)). In this case, there is no actual desulfurization, since oxidation is accomplished on one of the aromatic cores of DBT without the final product losing its sulfur atom. Likewise, there are microorganisms that can aerobically mineralize DBT (Kropp and Fedorak, Canad. J. Microbiol., 44, 605 (1998)). The use of these microorganisms for desulfurization purposes is not considered, because a significant loss of calorific power of the fuel thus treated would result therefrom.
The discovery of the strain Rhodococcus sp. IGTS8 (ATCC No. 53968) described in U.S. Pat. No. 5104801 allowed biodesulfurization to be considered as a conceivable proces that can be economically advantageous. This strain can aerobically remove sulfur from dibenzothiophene by specific oxidation of sulfur using the so-called 4S metabolic method (sulfoxide, sulfone, sulfinate, sulfite or sulfate). The final product resulting from desulfurization of DBT is 2-hydroxybiphenyl, and the sulfur is released in the form of sulfite (Oldfield et al., Microbiology, 143, 2961 (1997)). This new metabolic method was the subject of numerous studies. The DBT desulfurization phenotype is conferred by a dsz operon located on a plasmid. This operon codes for three enzymes, Dsz A, B and C, which are responsible for the oxidation reactions of DBT in hydroxybiphenyl (Li et al., J. Bacteriol., 178, 6409 (1996)). This operon was cloned and sequenced, and the metabolic method was described (Piddington et al., Appl. Environm. Microbiol., 61, 468 (1995)). A fourth enzyme, DszD, which acts to transport electrons, is also involved in this metabolism (Xi et al., Biochem. Biophys. Res. Commun., 230, 73 (1997)). These different enzymes have been purified and characterized (Gray et al., Nature Biotechnol., 14, 1705 (1996)). Genetic analysis revealed the existence of a promoter and activity regulation mechanisms. Thus, the expression of genes in the 4S method is suppressed by sulfur that is easily available such as sulfate, cysteine or methionine. Many patents have been filed on the use of the IGTS8 strain.
Since the isolation of the IGTS8 strain was described, several other groups of researchers reported the isolation of other strains able to use the 4S method by enrichment on a minimum mineral medium containing only DBT as a sulfur source. It is thus possible to cite Rhodococcus sp. SY1 (Omori et al., Biosci. Biotechnol. Bioeng., 59, 1195 (1995)) first described as being a Corynebacterium (Omori et al., Appl. Environm. Microbiol., 58, 911 (1992)), Rhodococcus erythropolis D-1 (Izumi et al., Appl. Environm. Microbiol., 60, 223 (1994)), Rhodococcus erythropolis H-2 (Ohshiro et al., FEMS Microbiol. Lett., 142, 65 (1996)), Rhodococcus UM3 and UM9 (Purdy et al., Curr. Microbiol., 27, 219 (1993)), Rhodococcus erythropolis (Wang and Krawiec, Arch. Microbiol., 161, 266 (1994)), Mycobacterium sp. strain G3 (Nekodzuka et al., Biocatal. Biotrans., 15, 17 (1997)), Paenibacillus sp. strain A11-1 and A11-2 (Konishi et al., Appl. Environm. Microbiol., 63, 3164 (1997)) which have the particular characteristic of being thermophilic, Arthrobacter paraffineus ECRD-1 (Lee et al., Appl. Environm. Microbiol., 61, 4362 (1995)) which was reclassified as actually being a Rhodococcus (Denis-Larose et al., Appl. Environm. Microbiol., 63, 2915 (1997)) and which was isolated on 4,6-diethyl dibenzothiophene, Arthrobacter sp. (E.P. 795603) which has the particular characteristic of acting on petroleum products without the addition of surfactants, Gordona CYSKI (Rhee et al., Appl. Environm. Microbiol., 64, 2327 1998), Sphingomonas sp. strain AD109 (PCT 98/45446).
Rhodococcus sp. IGTS8 has a number of properties which have made it particularly attractive for development in a biodesulfurization process (U.S. Pat. Nos. 5,104,801, 5,132,219, 5,198,341, 5,232,854, 5,344,778, 5,356,801, 5,356,813, 5,358,869, 5,358,870, 5,387,523, 5,472,875, 5,496,729, 5,510,265, 5,516,677, 5,529,930, 5,578,478, 5,733,773, 5,772,901, 5,811,285, . . . ). Implementation of such a process consists of several stages:
1) Cultivating the selected strain in a fermenter in the presence of carbon sources and other nutrients in such a way as to obtain the largest possible number of microorganism cells having the highest possible level of activity;
2) Harvesting these cells (separation of the bacterial biomass from the culture medium);
3) Using these cells in the form of xe2x80x9cresting cells,xe2x80x9d i.e., non-proliferating cells, in a biodesulfurization process during which enzymatic reactions take place that allow the organic sulfur of the feedstock to be treated to be transformed into sulfate or sulfite. This stage takes place in the presence of an aqueous phase;
4) Separating the different phases (oil, sulfate-loaded aqueous phase, biocatalyst solid phase);
5) Recycling all or part of the biocatalyst by adding new biocatalyst to it in such a way as to obtain sufficient activity;
6) Removing water from the desulfurized oil phase;
7) Removing sulfate from the aqueous phase.
To be used in an industrial process, however, any biodesulfurization biocatalyst must have sufficient stability.
Here, stability is defined as the stability of the biocatalyst during the biodesulfurization operation itself and not the stability of the strain (i.e., its preservation). From the economic standpoint, it is important to work in stage 3 with the most stable biocatalyst possible, with the price of the biocatalyst comprising a large part of the overall cost of the process. The stability, like the activity itself, is one of the key parameters in the viability of a biodesulfurization process. The cost of the process is directly proportional to it. Thus, a continuous biodesulfurization operation would only be seriously considered if a sufficiently stable biocatalyst were used. In addition, this also makes it possible to avoid the need to repeatedly add biocatalyst. In the case of a batch operation, the potential length of the operation and therefore the amount of substrate which will be treated will depend on the operating stability of the biocatalyst. It is known, however, that the strains that have been described for use in a biodesulfurization process have limited stability. Thus, strain IGTSB is described as having a half-life of 6 to 10 hours at 30xc2x0 C. (xe2x80x9cCommercial Development Progress Report and Market Update on Biocatalytic Desulfurization (BDS)xe2x80x9d, by J. A. Nagel presented at xe2x80x9cthe Catalytic Advances Program Meeting, Feb. 25-26, 1996). Half-life is defined as the time at the end of which the biocatalytic system has lost half its initial activity. Strains having desulfurization activity that are much more stable over time would allow substantial cost advantages.
Another patent such as WO 98 45446 describes the use of a strain of Sphingomonas to desulfurize fuels containing organosulfur-containing molecules. Another patent, WO 98 04678, describes the use of two strains of Rhodococcus, 213E and 213F, selected for their action on benzothiophene, whereby strain 213 F can desulfurize dibenzothiophene. Neither of these patents, however, describes or suggests a solution to the problem of the feasibility of using a biodesulfurization process on an industrial scale, i.e., the search for stable strains.
This invention is based on the discovery, isolation and use of new bacterial strains that can selectively attack the Cxe2x80x94S bonds of organic sulfur-containing molecules present in carbon products without altering the carbon structure of these molecules, and that would be stable enough to allow the aforementioned disadvantages to be avoided when using the strains in a biodesulfurization process.
Samples of these strains were submitted on May 20, 1999 to the National Collection of Microorganism Cultures of the Pasteur Institute (CNCM) under the numbers I-2204, I-2205, I-2206, I-2207, I-2208.
The strains described in this patent were isolated from soil samples taken from various coal storage sites. The isolation was performed by successive enrichment phases in a liquid medium and purification on a culture medium containing various sources of carbon (glycerol, glucose, succinate, ethanol) and dibenzothiophene as the only sulfur source. Special attention was focused on the composition of this medium to ensure that it would not contain any trace of sulfur other than the DBT. It is a specific minimum synthetic medium that does not contain any source of organic nitrogen and whose purity of components was selected such that the concentration of the sulfates in the medium is the lowest possible. From the various samples, fifteen or so pure strains that can use DBT as a single sulfur source were isolated.
We then eliminated the strains for which the product of degradation of DBT in the liquid medium was not hydroxybiphenyl (HBP). Thus, we kept only the strains that can degrade the DBT using the 4S method. To confirm this hypothesis and to evaluate the desulfurization activity (rate of disappearance of DBT by non-proliferating cells) of these strains, we cultivated them on the usual culture medium (minimum medium in the presence of DBT) in flasks, then we recovered the cells by centrifuging, and after washing with phosphate buffer and centrifuging, we used these cells in bioconversion as xe2x80x9cresting cellsxe2x80x9d on DBT. The reaction products were then analyzed by HPLC after addition of acetonitrile in the conversion medium. Concentrations of residual DBT and the HBP formed could thus be determined. Only the strains leading to an activity level greater than 1 mg of DBT degraded per gram of cells (dry weight) per hour were retained.
Thus having ten or so strains having DBT desulfurization activity using the 4S method, we selected the most stable strains. The stability of the activity of the strains was determined by incubation of xe2x80x9cresting cellsxe2x80x9d harvested after culturing the different strains under similar conditions. The initial activity of the cells was estimated using the conventional method. Then, the cells were incubated in the DBT-free conversion medium at 30xc2x0 C. while being stirred. The activity of cells after different incubation times at 30xc2x0 C. while being stirred was determined in the usual way after simple addition of DBT in the incubation medium to initiate the desulfurization reaction. This activity was then compared to the activity the cells had initially exhibited for the purpose of evaluating stability. This criterion is essential in terms of the process because it reflects the operating stability of the desulfurization system under pH and temperature conditions similar to operating conditions. These two parameters, however, are known to have a dramatic effect on the stability of enzymatic systems.
Based on this criterion, only five strains were selected. The selection criterion is based on residual DBT desulfurization activity that is at least equal to 10% of the initial activity after 96 hours of incubation in an aqueous medium at 30xc2x0 C. and at a pH of 7. Advantageously, the strains selected can have a residual DBT desulfurization activity after 96 hours of incubation in an aqueous medium at 30xc2x0 C. and at a pH of 7 of at least 30%, preferably at least 50%, most often at least 70%. Moreover, these strains have good stability in a two-phase water/oil medium, a medium that represents the desulfurization conditions of petroleum feedstocks.
These five pure strains have been characterized. They were subjected to a morphological examination by optical microscopy, using Gram staining as well as various biochemical tests.
The strains were then characterized by using them on API coryne galleries intended for identification of coryneform bacteria such as those described in this invention:
Based only on these characteristics, it was difficult to identify these strains precisely. That is why additional chemical-taxonomic analyses had to be performed. They consisted of an analysis of the composition of the bacterial walls and an overall analysis of the protein content of the cells.
Determination of the type of peptidoglycane did not reveal any major difference between the five strains, whereby the connecting diamino acid is meso-diaminopimelic acid (determination by thin-layer chromatography or TLC). The constituent sugars of the polymers of the wall are arabinose, galactose and glucose (TLC analysis). The five layers contain the same sugars and have no significant differences. They contain all five of the mycolates and the I-2206 strain differs in that it has one mycolic acid with a higher mobility (TLC analysis). The polar lipids are analyzed by thin layer chromatography which specifically shows phosphorylated lipids, aminated lipids and glycosylated lipids, as well as total lipids. The presence of phosphatidyl glycerol, phosphatidyl ethanolamine, and phosphatidyl inositol is also demonstrated in the five strains. Finally, these five strains have very similar peripheral characteristics. comparison with the databases reveals that they belong to the Rhodococcus/Gordona group. To refine the identification, an evaluation of the electrophoretic profile of the total proteins of each of the five strains was carried out (xe2x80x9csodium dodecyl sulfate polyacrylamide gel electroporesisxe2x80x9d or SDS-PAGE). The isolates were classified by digital analysis of the total protein profiles and comparison with the profile base. The four isolates I-2204, I-2205, I-2207 and I-2208 are included in the taxon Rhodococcus erythropolis, while I-2206 is grouped with the Rhodococcus rhodnii/Gordona cluster. As for I-2206, since it is more similar to database strains belonging to the Rhodococcus rhodnii species than to the Gordona strains referenced in the database, it appeared more logical to us to call it Rhodococcus rhodnii. 
More specifically, the invention relates to a process for desulfurization of a hydrocarbon feedstock containing organic sulfur-containing molecules using a bacterial culture in the presence of water and in an oxidizing atmosphere, at a temperature of between 20 and 40xc2x0 C., and at a pH of between 5 and 9, characterized in that it comprises the following stages:
a) The feedstock and the aqueous phase are brought into contact with the biological culture that comprises at least one bacterial strain formed by
Rhodococcus erythropolis CNCM I-2204
Rhodococcus erythropolis CNCM I-2205
Rhodococcus erythropolis CNCM I-2207
Rhodococcus erythropolis CNCM I-2208
Rhodococcus rhodnii CNCM I-2206
xe2x80x83or with its enzymatic derivatives, thereby obtaining a reaction medium;
b) a desulfurized oily phase that is recovered and an aqueous phase containing the biological culture are separated from the reaction medium, and
c) the aqueous phase in stage (a) is at least partially recycled.
The reaction medium may comprise an emulsified phase that comprises said biological culture, the aqueous phase and the oily phase, and said emulsified phase is separated from the reaction medium and recycled at least in part in stage a).
According to another characteristic, the aqueous phase may contain sulfates that can be eliminated using conventional techniques that are described, for example, above.
The desulfurization process using these strains may consist in bringing the strain or the enzymatic complex (crude extract) that has been isolated from any one of these strains into contact with the substrate that must be desulfurized in the presence of water. The amount of water present can be very low or high. It may be an emulsion or a non-emulsified two-phase mixture. In the case in which the amount of water is very low, it is possible for all of this water to be trapped by the cells and for the reaction medium to be assimilated into a suspension of cells in an organic medium (medium defined as microaqueous). The volumetric oil-to-water ratio can vary greatly. In the case of an emulsion, it can be obtained using surfactants or with mechanical systems. As a general rule, the mixture is brought to ambient temperature and atmospheric pressure.
In fact, any desulfurization process described can be used, since the improvement is caused by the biocatalyst itself which is more stable than the biocatalysts described in the prior art.
The desulfurization process described in this invention can be performed on crude oils or on fractions that are obtained from atmospheric distillation or vacuum distillation or on gasoils that are obtained from desulfurization units (HDS) to which it is desired to apply deep desulfurization, or on any other feedstock whose sulfur content must be reduced.
The desulfurization process in this invention can be carried out in batch mode or in fed-batch mode or continuously and includes the unitary operations described below:
1) Culture of bacteria
The biocatalysts are obtained by aerobic culture of the cells in a fermenter. The fermentation can be done in batch mode, in fed-batch mode or continuously. The culture medium contains at least one source of carbon that can be assimilated by the strains described in this document, at least one source of nitrogen and mineral salts. The carbon source can be, for example, glycerol provided in the form of industrial glycerine, ethanol, acetic acid or one of its salts, another organic acid or one of its salts, etc. The nitrogen source consists of ammonium salts or nitrate salts or urea. The culture medium can also contain sources of organic nitrogen, such as yeast extract, corn steep, peptones, etc. as long as their contribution does not cause an excess of sulfate in the culture medium. The mineral salts are present for the purpose of providing the potassium, sodium, magnesium, phosphorus, chlorine, iron, and calcium needed by the strains. The culture medium also contains at least one source of organic or mineral sulfur provided in the most conventional manner to ensure that synthesis of the enzymes responsible for desulfurization is not suppressed, i.e., by ensuring that the sulfate concentration in the culture medium is low, for example less than 50 mg/l. In this case, the organic sulfur source can be DMSO, DBT or any other compound containing organic sulfur molecules that can be assimilated by the strains described in this invention, for example a sulfur-containing gasoil fraction. In the case where culture medium contains too large a concentration of sulfate, for example greater than 50 mg/l, it will be necessary after their culture to incubate the cells in the presence of at least one source of organic sulfur such as dibenzothiophene, for example, before using them to trigger desulfurization activities.
Cells usually grow at temperatures of between 20 and 35xc2x0 C., preferably around 30xc2x0 C., while being stirred and aerated, and at a pH around neutral, 5 to 9 for example.
Several fermentation stages are most often necessary before creating the culture in the production fermenter; these stages constitute precultures. They make it possible to gradually progress from low-volume cultures to high-volume cultures. The ratio between the volume of inoculant and the volume of the culture under consideration is the inoculation rate (advantageously between 0.2 and 10%).
For example, the culture of strain CNCM I-2207 can be carried out according to the following protocol:
Precultures are created starting from a pure culture congelate of this strain. When the development of the preculture made it possible to obtain a sufficient biomass, corresponding to, for example, an optical density measured at 600 nm on the order of 5 per culture in batch mode on a medium identical to the one described in Example 1 below, the preculture or a portion of the latter is transferred in a sterile fashion to the biocatalyst production fermenter in such a way as to obtain an inoculation rate of 5%.
The production fermenter initially contains a liquid culture medium containing mineral salts and the usual vitamin supplements, avoiding any presence of sulfates, dimethyl sulfoxide (0.8 g/l) as a source of sulfur, a source of nitrogen (2 g/l of ammonium nitrate) and 5 g/l of sodium acetate as a source of carbon and energy. The concentration of this carbon source could be very low, even zero, if supply of the carbon substrate is begun promptly after inoculation. It should never exceed the inhibiting concentration, for example 15 g/l, in the case of acetate or acetic acid. Then the reactor is fed, preferably continuously, with a sterile aqueous solution containing a 5xc3x97 concentrated mineral medium (any combined component with the exception of the carbon source) and 6N acetic acid. This solution will be used as a pH regulating agent, whereby the latter is set at 7. Thus, the supply rate is automatically modulated in such a way as not to exceed a residual concentration of organic acid greater than the inhibiting concentration and not to have an acid concentration which remains at zero for long, since the demand for organic acid which is associated with the development of the strain is met by supplying a pH regulating agent. This is the principle of the pH-stat which is then advantageously applied. The fermentation medium is stirred (500 ppm) and aerated, and the temperature of the medium is kept under control in the range of 30xc2x0 C. Thus, after 90 hours of fermentation, the cell concentration is 18.5 g/l (dry weight) for a consumed carbon substrate of 61.4 g/l of acetic acid.
A carbon source can also be fed independently of the addition of a regulatory agent to ensure that it does not exceed the inhibiting acetic acid concentration, without for all that having a zero residual concentration of acid. It is also possible to continuously draw of f from the culture medium in order to bring about continuous fermentation with organic acid supply.
2) Preparation of the biocatalyst
After culturing in the fermenter, the cells are used as such (culture medium) or are harvested and separated from their culture medium by filtration, centrifuging or decanting, or any other means known to one skilled in the art. They can then optionally be subjected to special preparation treatments such as drying, freeze-drying, permeabilization, or immobilization so that they can be used in the biodesulfurization treatment.
Likewise, the enzymatic desulfurization system could be used instead of entire cells. In this case, an enzymatic extract will be prepared according to the usual methods, such as a simple crushing of cells, followed by centrifuging.
3) Biodesulfurization
The biodesulfurization reaction can be carried out in batch mode or in fed-batch mode or continuously, and in this latter case in a single stage or in a series of reactors. It is possible to use reactors of the air-lift type or stirred reactors that ensure both aeration of the medium (the reaction needs oxygen) and good dispersion of the different contributing phases.
The reaction can be carried out in a two-phase water/oil medium or in an emulsified medium. The volumetric water-to-oil ratio is variable and may fluctuate from 1/99 to 95/5. Water-to-oil ratios between 5/95 and 90/10, and most often between 20/80 and 80/20, are preferable.
The aqueous phase used can consist of at least in part of water that has been recovered after possible removal of sulfates. It can also be obtained from the microorganism culture medium.
The desulfurization reaction is usually carried out at a temperature of between 20 and 40xc2x0 C., preferably between 25 and 35xc2x0 C., and at a pH of between 5 and 9, preferably between 6 and 8, and at a pressure of generally between 1 and 3 bar (one bar=0.1 MPa).
The aqueous phase can contain an energy source in order to ensure physiological supply of electrons.
The biocatalyst used consists of any one of the forms described above. It can be obtained from a recycling operation after separation of the contributing phases.
All of these operations can be carried out continuously.
A continuous column reactor can also be used, whereby this reactor is first filled with biocatalyst in solid form, preferably immobilized on a solid support allowing good flow of the feedstock without clogging and ensuring that sufficient humidity is maintained (several %) around the biocatalyst so that it remains active when the feedstock to be treated passes. The feedstock can be passed through in countercurrent.
4) Separation of the phases
The conventional processes allowing separation of an oil phase from an aqueous phase can be applied. The following three phases result:
a) The desulfurized oil phase. The latter can optionally be dehydrated before use.
b) An aqueous phase containing the biocatalyst and optionally sulfates. The latter can optionally be re-used in the first or third stage after optional elimination of sulfates. Since the biocatalysts may be sensitive to sulfate content, the number of times the aqueous phase can be recycled will depend both on this sensitivity and on the concentration of sulfates generated by the biodesulfurization (that is, the sulfur content of the feedstock to be treated). Thus, the acceptable concentration of sulfates in this aqueous phase will depend on the strains used. By way of example, the desulfurization activity of strain I-2204 is not affected by sulfate concentrations equal to 5 g/l.
c) An emulsified phase containing biocatalyst cells in the presence of desulfurized oil and water. This phase, which can be directly recycled, can also be subjected to another separation stage (centrifuging, for example) or to another stage of washing with water, after which the oil and water phases obtained can be assimilated into the corresponding phases described above.
5) Elimination of sulfates
The aqueous phases obtained during the different stages described above are assembled before optional elimination of sulfates. The latter can be precipitated by the addition of elements that generate salts of insoluble sulfates. They can also be subjected to anaerobic biological treatment using sulfate-reducing microorganisms or to aerobic biological treatment.
This invention can be better understood, and its advantages will become more clear by reading the following examples. The latter are given as illustrative and nonlimiting examples of the invention. In particular, in these examples, the sulfur source used to reveal biodesulfurization activities is in all cases dibenzothiophene (DBT). In this case, this compound serves as a model molecule, but the strains described or cited in this invention can obviously be used in any process for biodesulfrization of petroleum feedstocks containing organic sulfur in the form of thiophene, benzothiophene, dibenzothiophene, naphthobenzothiophene, etc., and their derivatives.